Spinal Cord Neural Stem Cells Research Articles

Spinal cord neural stem cells derived from human embryonic stem cells promote synapse regeneration and remyelination in spinal cord injury model rats.

Spinal cord injury (SCI) is a devastating injury that significantly impairs patients' quality of life. To date, there is no effective treatment to mitigate nerve tissue damage and restore neurological function. Neural stem cells (NSCs) derived from human embryonic stem cells (hESCs) are considered an important cell source for reconstructing damaged neural circuits and enabling axonal regeneration. Recent preclinical studies have shown that NSCs are potential therapeutic cell sources for neuroprotection and neuroregeneration in SCI animal models. NSCs can be derived from different sources and the spinal cord-specific NSCs have a higher potential for the regeneration of SCI. However, the long-term therapeutic efficacy of spinal cord-specific NSCs remains unproven. Here, we generated human spinal cord NSCs (hSCNSCs) and investigated the effects of transplanted hSCNSCs on the repair of the SCI model rats for 60 days. The transplanted hSCNSCs improved BBB scores, reduced the lesion area and promoted an increase in the number of Nestin-positive cells in the spinal cord compared to the model rats. Meanwhile, hSCNSC transplantation promoted the expression of synaptophysin, a synaptic signature protein and MBP, a protein associated with remyelination. Interestingly, BAF45D, a chromatin remodelling factor that contributes to the induction of hSCNSCs with region-specific spinal cord identity, were increased by the hSCNSC transplantation. In addition, conditioned medium derived from the hSCNSCs also promoted regenerative repair of the injured spinal cord. These results demonstrate that hSCNSCs may play a critical role in the regenerative repair of SCI.

Therapeutic potential of EVs loaded with CB2 receptor agonist in spinal cord injury via the Nrf2/HO-1 pathway

ABSTRACT Background Spinal cord injury (SCI) poses a challenge due to limited treatment options. Recently, the effect and mechanism of Exo-loaded cannabinoid receptor type 2 (CB2) agonist AM1241(Exo + AM1241) have been applied in other inflammatory diseases but not in SCI. Methods The SCI model was set up using C57BL/6 mice, followed by the treatment of Exo, AM1241, and Exo + AM1241. We assessed the effects of the following treatments on motor function recovery using BMS, and evaluated histological changes, apoptosis activity, inflammation, and oxidative stress in the SCI mice model. Additionally, the effect of following treatments on spinal cord neural stem cells (NSCs) was evaluated under lipopolysaccharides (LPS) induced inflammatory and oxidative models and, glutamate (Gluts) induced cell apoptosis models. Result Our results demonstrated that Exo + AM1241 treatment significantly improved motor function recovery, after SCI by decreasing proinflammatory cytokines, and suppressing astrocyte/microglia (GFAP/Iba1) activation in the injury zone. Additionally, this treatment reduces pro-apoptotic proteins (Bax and caspase 3), increases the levels of the anti-apoptotic protein Bcl-2, enhances antioxidant defenses by boosting SOD and GSH, and lowers oxidative stress markers such as MDA. It also activates the Nuclear factor erythroid-2 (Nrf2) related factor 2 signaling pathway, thereby enhancing tissue protection against damage and cell death.

An improved method for generating human spinal cord neural stem cells

Neural stem cells have exhibited efficacy in pre-clinical models of spinal cord injury (SCI) and are on a translational path to human testing. We recently reported that neural stem cells must be driven to a spinal cord fate to optimize host axonal regeneration into sites of implantation in the injured spinal cord, where they subsequently form neural relays across the lesion that support significant functional improvement. We also reported methods of deriving and culturing human spinal cord neural stem cells derived from embryonic stem cells that can be sustained over serial high passage numbers in vitro, providing a potentially optimized cell source for human clinical trials. We now report further optimization of methods for deriving and sustaining cultures of human spinal cord neural stem cell lines that result in improved karyotypic stability while retaining anatomical efficacy in vivo. This development improves prospects for safe human translation.

BAF45D-binding to HOX genes was differentially targeted in H9-derived spinal cord neural stem cells

Chromatin accessibility has been used to define how cells adopt region-specific neural fates. BAF45D is one of the subunits of a specialised chromatin remodelling BAF complex. It has been reported that BAF45D is expressed in spinal cord neural stem cells (NSCs) and regulates their fate specification. Within the developing vertebrate spinal cord, HOX genes exhibit spatially restricted expression patterns. However, the chromatin accessibility of BAF45D binding HOX genes in spinal cord NSCs is unclear. In the present study, we found that in H9-derived spinal cord NSCs, BAF45D targets TBX6, a gene that regulates spinal cord neural mesodermal progenitors. Furthermore, BAF45D binding to the NES gene is much more enriched in H9-derived spinal cord NSCs chromatin compared to ESCs chromatin. In addition, BAF45D binding to anterior and trunk/central HOX genes, but not to lumbosacral HOX genes, was much more enriched in NSCs chromatin compared to ESCs chromatin. These results may shed new light on the role of BAF45D in regulating region-specific spinal cord NSCs by targeting HOX genes.

In-vitro differentiation of human embryonic stem cells into spinal cord neural stem cells.

In-vitro differentiation of human embryonic stem cells into spinal cord neural stem cells (NSCs) can help researchers better understand the cellular processes associated with spinal cord development and regeneration, and provide therapeutic strategies for spinal cord disorders. However, effective and consistent methods for the generation of human spinal cord NSCs are rare. Objective of the study is to establish methods for the in-vitro induction and long-term maintenance of human spinal cord NSCs. H9 cells were treated with neural induction medium for 10 days under single-cell seeding condition, followed by treatment with neural maintenance medium and replacement with NSC medium after five passages. The identity of the generated cells was determined by immunofluorescence, immunoblotting, and cleavage under targets and tagmentation (CUT&Tag) assays. After the neural induction, OCT4, an embryonic stem cell marker, was significantly reduced, whereas NESTIN and PAX6, two NSC markers, were clearly increased. After the neural maintenance, most of the H9-derived cells consistently expressed NESTIN and PAX6 together with SOX1 and HOXC9, two spinal cord markers. The Homer known motif enrichment results of the CUT&Tag assay confirmed the expression of HOXC9 in the H9-derived spinal cord NSCs, which can be maintained for more than 40 days under an in vitro culture system. This study sheds new light on effective induction and maintenance of human spinal cord NSCs.

Sulfation of Glycosaminoglycans Modulates the Cell Cycle of Embryonic Mouse Spinal Cord Neural Stem Cells.

In the developing spinal cord neural stem and progenitor cells (NSPCs) secrete and are surrounded by extracellular matrix (ECM) molecules that influence their lineage decisions. The chondroitin sulfate proteoglycan (CSPG) DSD-1-PG is an isoform of receptor protein tyrosine phosphatase-beta/zeta (RPTPβ/ζ), a trans-membrane receptor expressed by NSPCs. The chondroitin sulfate glycosaminoglycan chains are sulfated at distinct positions by sulfotransferases, thereby generating the distinct DSD-1-epitope that is recognized by the monoclonal antibody (mAb) 473HD. We detected the epitope, the critical enzymes and RPTPβ/ζ in the developing spinal cord. To obtain insight into potential biological functions, we exposed spinal cord NSPCs to sodium chlorate. The reagent suppresses the sulfation of glycosaminoglycans, thereby erasing any sulfation code expressed by the glycosaminoglycan polymers. When NSPCs were treated with chlorate and cultivated in the presence of FGF2, their proliferation rate was clearly reduced, while NSPCs exposed to EGF were less affected. Time-lapse video microscopy and subsequent single-cell tracking revealed that pedigrees of NSPCs cultivated with FGF2 were strongly disrupted when sulfation was suppressed. Furthermore, the NSPCs displayed a protracted cell cycle length. We conclude that the inhibition of sulfation with sodium chlorate interferes with the FGF2-dependent cell cycle progression in spinal cord NSPCs.

Valproic Acid Labeled Chitosan Nanoparticles Promote the Proliferation and Differentiation of Neural Stem Cells After Spinal Cord Injury.

Chitosan nanoparticles and valproic acid are demonstrated as the protective agents in the treatment of spinal cord injury (SCI). However, the effects of valproic acid-labeled chitosan nanoparticles (VA-CN) on endogenous spinal cord neural stem cells (NSCs) following SCI and the underlying mechanisms involved remain to be elucidated. In this study, the VA-CN was constructed and the effects of VA-CN on NSCs were assessed in a rat model of SCI. We found VA-CN treatment promoted recovery of the tissue and locomotive function following SCI. Moreover, administration of VA-CN significantly enhanced neural stem cell proliferation and the expression levels of neurotrophic factors following SCI. Furthermore, administration of VA-CN led to a decrease in the number of microglia following SCI. In addition, VA-CN treatment significantly increased the Tuj 1- positive cells in the spinal cord of the SCI rats, suggesting that VA-CN could enhance the differentiation of NSCs following SCI. In conclusion, these results demonstrated that VA-CN could improve the functional and histological recovery through promoting the proliferation and differentiation of NSCs following SCI, which would provide a newly potential therapeutic manner for the treatment of SCI.

Fabrication of homotypic neural ribbons as a multiplex platform optimized for spinal cord delivery

Cell therapy for the injured spinal cord will rely on combined advances in human stem cell technologies and delivery strategies. Here we encapsulate homotypic spinal cord neural stem cells (scNSCs) in an alginate-based neural ribbon delivery platform. We perform a comprehensive in vitro analysis and qualitatively demonstrate graft survival and injury site retention using a rat C4 hemi-contusion model. Pre-configured neural ribbons are transport-stable modules that enable site-ready injection, and can support scNSC survival and retention in vivo. Neural ribbons offer multifunctionality in vitro including co-encapsulation of the injury site extracellular matrix modifier chondroitinase ABC (chABC), tested here in glial scar models, and ability of cervically-patterned scNSCs to differentiate within neural ribbons and project axons for integration with 3-D external matrices. This is the first extensive in vitro characterization of neural ribbon technology, and constitutes a plausible method for reproducible delivery, placement, and retention of viable neural cells in vivo.

Spinal cord neural stem cells heterogeneity in postnatal development

Neural stem cells are capable of generating new neurons during development as well as in the adulthood and represent one of the most promising tools to replace lost or damaged neurons after injury or neurodegenerative disease. Unlike the brain, neurogenesis in the adult spinal cord is poorly explored and the comprehensive characterization of the cells that constitute stem cell neurogenic niche is still missing. Moreover, the terminology used to specify developmental and/or anatomical CNS regions, where neurogenesis in the spinal cord occurs, is not consensual and the analogy with the brain is often unclear. In this review, we will try to describe the heterogeneity of the stem cell types in the spinal cord ependymal zone, based on their origin and stem cell potential. We will also consider specific animal in vitro models that could be useful to identify “the right” stem cell candidate for cell replacement therapies.

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes.

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) throughout life, which proliferate to reform the ependymal tube and differentiate into lost neurons after spinal cord injury. Deciphering how these NSCs retain pluripotency post-development and proliferate upon spinal cord injury to reform the exact pre-injury structure can provide valuable insight into how mammalian spinal cords may regenerate as well as potential treatment options. Performing gene knock-outs in specific subsets of NSCs within a restricted time period will allow study of the molecular mechanisms behind these regenerative processes, without being confounded by development perturbing effects. Described here is a method to perform gene knock-out in axolotl spinal cord NSCs using the CRISPR-Cas9 system. By injecting the CAS9-gRNA complex into the spinal cord central canal followed by electroporation, target genes are knocked out in NSCs within specific regions of the spinal cord at a desired timepoint, allowing for molecular studies of spinal cord NSCs during regeneration.

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

Generation and post-injury integration of human spinal cord neural stem cells.

Spinal cord neural stem cells (NSCs) have great potential to reconstitute damaged spinal neural circuitry, but they have yet to be generated in vitro. We now report the derivation of spinal cord NSCs from human pluripotent stem cells (hPSCs). Our observations show that these spinal cord NSCs differentiate into a diverse population of spinal cord neurons occupying multiple positions along the dorso-ventral axis, and can be maintained for prolonged time periods. Grafts into injured spinal cords were rich with excitatory neurons, extended large numbers of axons over long distances, innervated their target structures, and enabled robust corticospinal regeneration. The grafts synaptically integrated into multiple host intraspinal and supraspinal systems, including the corticospinal projection, and improved functional outcomes after injury. hPSC-derived spinal cord NSCs could enable a broad range of biomedical applications for in vitro disease modeling and constitute an improved clinically translatable cell source for 'replacement' strategies in several spinal cord disorders.

P.089 AMPA receptor modulation as a therapeutic strategy to enhance survival of spinal cord neural stem cells

Background: Transplantation of neural stem/progenitor cells (NSPCs) following spinal cord injury (SCI) is a promising strategy to enhance regeneration but is limited by poor survival of grafted cells. Recently, we demonstrated for the first time that the excitatory neurotransmitter glutamate, which is released after SCI, promotes survival/proliferation of spinal cord NSPCs via the AMPA subtype of glutamate receptors. Here, we examine the therapeutic potential of selective AMPA receptor modulation on NSPC survival using allosteric AMPA receptor modulators known as ampakines. Methods: NSPCs from the periventricular region of the adult rat spinal cord were treated with ampakines CX614 and CX546 for 72h either alone or in the presence of low-dose glutamate (50uM). Results: Treatment with CX-546 or CX-614 in the presence of glutamate led to a significant increase in live cell numbers. This was due to both a reduction in cell death and increase in cellular proliferation. Ampakine/glutamate treatment led to a significant increase in cell survival compared to controls in the setting of oxidative stress. Conclusions: We present the first examination of the effect of allosteric AMPA receptor modulators on adult spinal cord--derived NSPCs. Positive modulation of AMPA receptors may be a promising therapeutic strategy in the subacute/chronic phases after SCI to increase survival of endogenous or transplanted NSPCs.

Selective killing of spinal cord neural stem cells impairs locomotor recovery in a mouse model of spinal cord injury

BackgroundSpinal cord injury (SCI) is a devastating condition mainly deriving from a traumatic damage of the spinal cord (SC). Immune cells and endogenous SC-neural stem cells (SC-NSCs) play a critical role in wound healing processes, although both are ineffective to completely restore tissue functioning. The role of SC-NSCs in SCI and, in particular, whether such cells can interplay with the immune response are poorly investigated issues, although mechanisms governing such interactions might open new avenues to develop novel therapeutic approaches.MethodsWe used two transgenic mouse lines to trace as well as to kill SC-NSCs in mice receiving SCI. We used Nestin CreERT2 mice to trace SC-NSCs descendants in the spinal cord of mice subjected to SCI. While mice carrying the suicide gene thymidine kinase (TK) along with the GFP reporter, under the control of the Nestin promoter regions (NestinTK mice) were used to label and selectively kill SC-NSCs.ResultsWe found that SC-NSCs are capable to self-activate after SCI. In addition, a significant worsening of clinical and pathological features of SCI was observed in the NestinTK mice, upon selective ablation of SC-NSCs before the injury induction. Finally, mice lacking in SC-NSCs and receiving SCI displayed reduced levels of different neurotrophic factors in the SC and significantly higher number of M1-like myeloid cells.ConclusionOur data show that SC-NSCs undergo cell proliferation in response to traumatic spinal cord injury. Mice lacking SC-NSCs display overt microglia activation and exaggerate expression of pro-inflammatory cytokines. The absence of SC-NSCs impaired functional recovery as well as neuronal and oligodendrocyte cell survival. Collectively our data indicate that SC-NSCs can interact with microglia/macrophages modulating their activation/responses and that such interaction is importantly involved in mechanisms leading tissue recovery.

Bridging the gap with functional collagen scaffolds: tuning endogenous neural stem cells for severe spinal cord injury repair.

Severe spinal cord injury (SCI) induces massive proliferation of spinal cord neural stem cells (NSCs), which are considered a promising cell source for therapeutic neural repair. However, most injury-activated spinal cord NSCs differentiate into astrocytes, rather than neurons, in the lesion site as a result of the injury-derived microenvironment. In this mini-review, we introduce the concept of tuning injury-activated endogenous NSCs by implantation of functionalized collagen scaffolds for severe SCI repair. The current state of in situ modulation of migration, scar formation, neuronal differentiation, and functional integration of injury-activated NSCs and their progeny by implantation of elaborately modified collagen scaffolds is demonstrated. Moreover, challenges and perspectives for utilizing injury-activated NSCs for regenerative repair of severe SCI are also discussed.

Chemically defined differentiation of human pluripotent stem cells to hindbrain and spinal cord neural stem cells with defined regional identities

Chemically defined differentiation of human pluripotent stem cells to hindbrain and spinal cord neural stem cells with defined regional identities

Endogenous neural stem cell responses to stroke and spinal cord injury.

Stroke and spinal cord injury (SCI) are among the most frequent causes of central nervous system (CNS) dysfunction, affecting millions of people worldwide each year. The personal and financial costs for affected individuals, their families, and the broader communities are enormous. Although the mammalian CNS exhibits little spontaneous regeneration and self-repair, recent discoveries have revealed that subpopulations of glial cells in the adult forebrain subventricular zone and the spinal cord ependymal zone possess neural stem cell properties. These endogenous neural stem cells react to stroke and SCI by contributing a significant number of new neural cells to formation of the glial scar. These findings have raised hopes that new therapeutic strategies can be designed based on appropriate modulation of endogenous neural stem cell responses to CNS injury. Here, we review the responses of forebrain and spinal cord neural stem cells to stroke and SCI, the role of these responses in restricting injury-induced tissue loss, and the possibility of directing these responses to promote anatomical and functional repair of the CNS.

EphB/ephrin-B signaling controls continuous proliferation of the adult spinal cord neural stem cells

EphB/ephrin-B signaling controls continuous proliferation of the adult spinal cord neural stem cells

A Mesenchymal-Like ZEB1+ Niche Harbors Dorsal Radial Glial Fibrillary Acidic Protein-Positive Stem Cells in the Spinal Cord

In humans and rodents the adult spinal cord harbors neural stem cells located around the central canal. Their identity, precise location, and specific signaling are still ill-defined and controversial. We report here on a detailed analysis of this niche. Using microdissection and glial fibrillary acidic protein (GFAP)-green fluorescent protein (GFP) transgenic mice, we demonstrate that neural stem cells are mostly dorsally located GFAP(+) cells lying ependymally and subependymally that extend radial processes toward the pial surface. The niche also harbors doublecortin protein (Dcx)(+) Nkx6.1(+) neurons sending processes into the lumen. Cervical and lumbar spinal cord neural stem cells maintain expression of specific rostro-caudal Hox gene combinations and the niche shows high levels of signaling proteins (CD15, Jagged1, Hes1, differential screening-selected gene aberrative in neuroblastoma [DAN]). More surprisingly, the niche displays mesenchymal traits such as expression of epithelial-mesenchymal-transition zinc finger E-box-binding protein 1 (ZEB1) transcription factor and smooth muscle actin. We found ZEB1 to be essential for neural stem cell survival in vitro. Proliferation within the niche progressively ceases around 13 weeks when the spinal cord reaches its final size, suggesting an active role in postnatal development. In addition to hippocampus and subventricular zone niches, adult spinal cord constitutes a third central nervous system stem cell niche with specific signaling, cellular, and structural characteristics that could possibly be manipulated to alleviate spinal cord traumatic and degenerative diseases.

Superparamagnetic iron oxide labeling of spinal cord neural stem cells genetically modified by nerve growth factor-β

This study established superparamagnetic iron oxide (SPIO)-labeled nerve growth factor-beta (NGF-beta) gene-modified spinal cord-derived neural stem cells (NSCs). The E14 rat embryonic spinal cord-derived NSCs were isolated and cultured. The cells of the third passage were transfected with plasmid pcDNA3-hNGFbeta by using FuGENE HD transfection reagent. The expression of NGF-beta was measured by immunocytochemistry and Western blotting. The positive clones were selected, allowed to proliferate and then labeled with SPIO, which was mediated by FuGENE HD transfection reagent. Prussian blue staining and transmission electron microscopy (TEM) were used to identify the SPIO particles in the cells. The distinctive markers for stem cells (nestin), neuron (beta-III-tubulin), oligodendrocyte (CNPase) and astrocyte (GFAP) were employed to evaluate the differentiation ability of the labeled cells. The immunocytochemistry and western blotting showed that NGF-beta was expressed in spinal cord-derived NSCs. Prussian blue staining indicated that numerous blue-stained particles appeared in the cytoplasma of the labeled cells. TEM showed that SPIO particles were found in vacuolar structures of different sizes and the cytoplasma. The immunocytochemistry demonstrated that the labeled cells were nestin-positive. After differentiation, the cells expressed beta-III-tubulin, CNPase and GFAP. It was concluded that the SPIO-labeled NGF-beta gene-modified spinal cord-derived NSC were successfully established, which are multipotent and capable of self-renewal.